Our data establish three new and distinct but inter-related signaling mechanisms: the mutual dependence and synergistic interaction between the Gq-PLC-β and Akt-WNK1 signaling pathways; the regulation of PLC-β activity by modulating the availability of its substrate; and the regulation of PI4KIIIα by WNK1 to control the concentration of PIP2-derived second messengers.
As summarized in , blocking WNK1 activity markedly inhibits PLC-β signaling by Gq-coupled receptors and, conversely, inhibiting Gq blocks PLC-β signaling by WNK1. Inhibition could be exerted by several approaches and at several steps in each pathway, both in cells and in detached membrane patches. In each case, replenishment of the inhibited or depleted species, or stimulation of a downstream component, restored normal activities of both WNK1 and Gq-PLC-β. Importantly, WNK1 can elicit PLC signals without added GPCR agonist, and WNK1 synergistically amplifies the stimulation of PLC-β by Gq.
In contrast to G proteins, WNK1 increases PLC-β activity by increasing the supply of the PIP2
substrate. Because the concentration of PIP2
in the plasma membrane is below Km
], PLC-β displays first-order substrate kinetics and increase in available PIP2
linearly amplifies PLC-β output. Note that WNK1 only modulates Gq
PLC-β. Other outputs, such as stimulation of a rho GEF [36
], are not involved. WNK1 thus alters the spectrum of Gq
outputs as well as its amplitude.
WNK1 promotes synthesis of PIP2
by somehow activating PI4KIIIα ( and ). PI4KIIIα is important for the re-synthesis of PIP2
following hydrolysis by receptor-stimulated PLC [30
], but how the activity of PI4KIIIα is regulated is unknown. The amount of PI4KIIIα is decreased in membranes from WNK1 knockdown cells, suggesting that regulation of PI4KIIIα by WNK1 may involve its redistribution between plasma membrane and intracellular pools. However, WNK1 must do more than just recruit PI4KIIIα to the plasma membrane because it remains necessary for PLC-β signaling in detached patches. We have no evidence that WNK1 either binds or phosphorylates PI4KIIIα, and additional factors may be involved.
Regardless, our results indicate that the activity of PI4KIIIα is regulated and that its regulation by WNK1 is critical for PLC-β signaling by Gq-coupled receptors. The basal activity of WNK1 provides adequate tonic stimulation of PI4KIIIα to provide PIP2 for normal PLC-β signaling. However, additional WNK1 markedly amplifies signaling by Gq-coupled receptor even at a concentration that does not by itself activate TRPC6 (). In addition, PI3K-activating growth factors such as IGF1 activate WNK1 to further potentiate Gq-PLC-β signaling, probably via Akt-mediated phosphorylation of WNK1 at T58 (). These results suggest that WNK1 is an important physiological mediator of the Akt kinase signaling pathway and coordinates Akt signaling with the Gq-PLC-β signaling pathway.
This action of WNK1 represents the first known instance of acute control of PIP2
availability as a mechanism of regulation of cellular DAG and IP3
concentrations without concurrent stimulation of the intrinsic activity of PLC. Application of WNK1 alone stimulates the production of DAG measured using the TRPC6 biosensor by an amount comparable to that initiated by GTPγS (). Some low output of PLC-β, evidently dependent on basal input from Gαq
, is adequate to support this WNK1 signaling. By promoting PIP2
synthesis, WNK1 can also acutely modulate the amplitude of receptor-stimulated PLC-β signaling and/or its duration. Inhibition of endogenous WNK1 by anti-WNK1 antibody blocks both the DAG and IP3
limbs of PLC-β signaling stimulated by multiple Gq
-coupled receptors by ≥50% ( and Figures S1D–F and S3
). Acute stimulation of WNK1 activity by IGF1 potentiates PLC-β signaling by ~2-fold (). WNK1 thus controls a major mode of desensitization of Gq
signaling in addition to modulating acute responses. PIP2
itself is a signaling molecule that regulates many plasma membrane targets [37
]. Increasing PIP2
synthesis by WNK1 is thus an important mechanism for increasing cellular DAG and IP3
concentrations without causing untoward effects of decreases in membrane PIP2
on other targets.
PLC-β enzymes function as co-incidence detectors that synergistically integrate multiple upstream inputs including Gαq
, Gβγ, Ca2+
, and Rac [35
]. These mechanisms of synergism are results of combined super-additive stimulation of the catalytic activity of PLC-β. In contrast, synergism between WNK1 and Gαq
depends on the ability of WNK1 to enhance substrate supply for PLC-β. This system is analogous to the interaction between PLCs and inositol phosphate kinases in which PLC provides the IP3
starting material that the cell-type-specific kinases then phosphorylate to the appropriate inositol polyphosphates [40
The interactive control of PLC-β by WNK and Gq
may have important implications in the pathogenesis of hypertension in patients with PHA2 that is caused by mutations of WNK1
that increase WNK1 expression [2
-mediated PLC-β signaling plays a central role in the regulation of vascular tone by virtually all vasoactive hormones [41
], and potentiation of PLC-β signaling by WNK1 would promote vasoconstriction and cause hypertension. The role of WNK1 in the regulation of vascular tone is supported by the report that mice with heterozygous inactivation of Wnk1
display reduced blood pressure without renal Na+
wasting and hypovolemia [42
]. Finally, the similarity between the phenotypes of WNK1-knockout and Gαq
-knockout mice (both lethal at embryonic day ~10.5, with cardiac defects) supports the idea that interaction between the WNK1 and Gαq/11
pathways is important for embryonic development [10
In conclusion, WNK1 potentiates Gq-PLC-β signaling by stimulating the activity of PI4KIIIα to increase synthesis of PIP2. Phosphorylation of WNK1 by Akt kinase increases its activity in this pathway, and allows tyrosine kinase receptors or other activators of the Akt kinase cascade to regulate G protein-mediated PLC-β signaling. The extent and physiological role of WNK1 in the regulation of PLC-β signaling will probably differ among specialized cells. Our understanding of these interactions should expand as we learn more ways in which WNK1 activity is regulated.